Abstract Although ?-synuclein (?-syn) is largely a cytosolic protein, extracellular ?-syn is believed to play an important role in the pathology of PD, contributing to key processes such as the progressive, prion-like spread of Lewy body pathology throughout the brain, and initiation of cellular pathology in neurons and glia. In neuronal systems, ?-syn was found to be exported through a non-classical pathway and secreted in extracellular vesicles (EVs). In addition to its implications for understanding the role of ?-syn in PD pathogenesis, the localization of ?-syn to EVs presents a unique strategy for utilizing peripheral ?-syn as a PD biomarker, by specifically targeting ?-syn in EVs originating in the brain. Indeed, we have recently demonstrated that ?-syn crosses from brain to blood, and that a fraction of this central nervous system (CNS)-derived ?-syn, recovered from plasma, is contained within exosomes (small EVs of endocytic origin). Further, this peripherally accessible exosomal ?-syn performed similarly to cerebrospinal fluid (CSF) total ?-syn in differentiating control from PD human subjects. Intriguingly, while CSF ?-syn is decreased in PD, CNS-derived exosomal ?-syn was increased, suggesting that clearance of ?-syn from the brain in EVs may be up-regulated in PD. However, only a small portion of intracerebroventricularly-injected ?-syn was associated with blood exosomes; the compartment in which the rest resided is not known. For example, it is unclear whether shedding microvesicles (MVs, vesicles that bud directly from the plasma membrane) are involved in ?-syn secretion, especially from the perspective of CNS-derived plasma EVs as PD biomarkers, nor is it clear what types of cells may participate in generating ?-syn-containing vesicles that cross the BBB. Finally, the role of ?-syn secretion via EVs in PD pathogenesis remains to be defined. Here, we propose to investigate whether ?-syn transported from the brain to the blood via EVs potentially provides a mechanism for clearance of toxic ?-syn species, as well as a potential cellular source of EV-contained ?-syn altered in PD patients, which may be suitable for use as diagnostic or progression biomarkers. We will first examine the total and post-translationally modified (phosphorylated and oligomeric) ?-syn in plasma EVs derived from neurons, astrocytes, and oligodendrocytes. This protein will be characterized both using sensitive, quantitative immunoassays, as well as Nanoparticle Tracking Analysis (NTA), to determine the distribution of ?-syn forms in different types of EVs. Both types of analysis will be used to compare samples from PD patients, healthy controls, and patients with multiple system atrophy, a related but distinct synucleinopathy featuring oligodendrocyte inclusions. We will then develop an animal model suitable for studying the mechanisms by which EV-contained ?-syn may cross the BBB and enter the plasma, and begin to study the potential mechanisms of ?-syn-containing EV transport across the BBB. These experiments will be valuable for future studies aimed at developing biomarkers for PD progression, as well as for seeking novel therapeutic treatments aimed at this potential clearance mechanism.